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EGFL6 Promotes Bone Metastasis of Lung Adenocarcinoma by Enhancing Osteoclast Differentiation and Stimulating Cancer Cell Metastasis

Lung cancer is still the most fatal cancer today with approximately 30%-40% of these patients will develop bone metastasis. Lung adenocarcinoma (LUAD) is the most common and aggressive type of lung cancer. The relationship between LUAD and bone metastasis and its underlying mechanism remains unclear. This study proved that epidermal growth factor-like domain multiple 6 (EGFL6) was highly expressed in LUAD specimens of patients, and the expression level was positively correlated with bone metastasis of LUAD. Wnt/β-catenin 37 PI3K/AKT/mTOR 38 , MAPK 39 , Notch 40 In Wnt/β-catenin β-catenin glycogen synthase (GSK3), and polyposis (APC). Phosphorylation GSK3 The effect of EGFL6 on EMT markers (E-cadherin, N-cadherin and Vimentin) in A549 detected by ICC. Over-expression of EGFL6 increases the expression of N-cadherin and Vimentin in cytoplasm but attenuates the expression of E-cadherin while knockdown of EGFL6 displays the adverse. b Western blotting analysis is utilized to detect the levels of p-GSK-3, GSK-3 and β-catenin under the inuence of over-expression and knockdown of EGFL6 in A549 cells. c Western blotting analysis is utilized to detect the levels of p-PI3K, PI3K, p-AKT, AKT, p-mTOR and mTOR under the inuence of over-expression and knockdown of EGFL6 in A549 cells. Results were presented as mean ± SD. P <0.05 was considered statistically signicant. * P <0.05, ** P <0.01, *** P <0.001.

Results
EGFL6 is elevated in LUAD and is associated with bone metastasis. In vivo, implantation of human adenocarcinoma A549 cells with a higher expression of EGFL6 not only increased tumor growth rate but also bone resorption of tibias in nude mice. In vitro, the secretion of EGFL6 from A549 cells increased osteoclast differentiation but had little effect on osteogenic differentiation. To reveal the underlying mechanism, we demonstrated that EGFL6 enhanced osteoclast differentiation through activating nuclear factor-kappa B (NF-κB) and downstream c-Fos/NFATc1 signaling pathways, and in addition promoted the proliferation, migration and invasion of A549 cells through enhancing the epithelial mesenchymal transformation (EMT) and promoting Wnt/β-catenin and PI3K/AKT/mTOR signaling pathways.

Conclusions
We unveil EGFL6 as a predictor in bone metastasis of LUAD and underscore the relevance of EGFL6 as a therapeutic target.

Background
Lung cancer is the leading cause of cancer deaths worldwide, among which lung adenocarcinoma (LUAD) is the most common and aggressive type 1, 2 . The skeletal system is one of the most common metastatic sites in lung cancer patients, accounting for approximately 30%-40% of lung cancer patients 3, Page 4/22 4 . Bone metastasis patients often suffer skeletal-related events (SREs) including pathologic fractures, spinal cord compression, bone marrow aplasia, and hypercalcemia 5 . Among all types of lung cancer, LUAD has the highest incidence of bone metastases 6 . However, the underlying mechanism of bone metastasis in lung cancer, especially in LUAD, remains unclear. Studies were carried out to identify the interaction of lung cancer cells with bone marrow cells. Nakamura ES et al demonstrated that osteoclasts might promote bone metastasis of lung cancer cells by producing macrophage-derived chemokine CCL22, which upregulated receptor activator of the NF-κB ligand (RANKL) in osteoclast-like cells 7 . The expression of bone remodeling cytokines or receptors, such as RANKL, receptor activator of NF-κB (RANK), and osteoprotegerin (OPG), were found to be upregulated in bone metastatic tissues from primary non-small cell lung cancer (NSCLC) patients. In addition, the increased expression of RANKL and OPG was correlated with clinical features of the patients 8 . As a collagen receptor, Discoidin domain receptor-1 (DDR1) was found to associate with poor clinical outcome in lung cancer. In a nude mice model of tumor with bone metastasis, the lack of DDR1 showed decreased metastatic activity and osteolytic lesions 9 . A representative study determined that the activation of the WNT/TCF signaling pathway mediates the metastasis of LUAD to bones and brain 10 . However, the signaling pathway regulates the LUAD metastasis is still largely unknown.
Tumor growth and metastasis require the formation of new blood vessels, which is termed angiogenesis. Epidermal growth factor-like domain multiple 6 (EGFL6) is an exocrine protein that plays an important role in promoting endothelial cell migration and angiogenesis 11 . Ectopic expression of EGFL6 has been shown to promote the invasion of cancer cells in breast, gastric, and nasopharyngeal cancers [12][13][14] . In addition, cumulated evidence proved that EGFL6 exerted a pivot role in the proliferation, migration, and invasion of several tumor cells, such as breast, ovarian, and colorectal cancer cells 15,16 . Clinically, immunohistochemical staining of 150 LUAD specimens showed that high EGFL6 expression was associated with poor prognosis of LUAD, especially in young patients (<69 years) 17 . However, this study focused on the relationship between the survival rate and the expression level of EGFL6 in LUAD. It has not been studied whether there is a relationship between the expression of EGFL6 and the occurrence of bone metastasis.
Here, we proved that EGFL6 was highly expressed in LUAD samples and was positively correlated with bone metastasis and TNM stage. We demonstrated that EGFL6 enhanced osteoclast differentiation and bone resorption through activating NF-κB and downstream c-Fos/NFATc1 signaling pathways but exerted little effect on the formation of osteoblasts. Additionally, EGFL6 also promoted the proliferation, migration and invasion of LUAD cells through epithelial mesenchymal transformation (EMT) and Wnt/βcatenin and PI3K/AKT/mTOR signaling pathways. Therefore, we propose that EGFL6 can not only be used as a predictor of the prognosis of LUAD, but also play a role in promoting LUAD bone metastasis and subsequent bone destruction.

Specimen collection
We collected 30 LUAD tissues and matched adjacent normal tissues from the Biological Resource Center of Enze Medical Center, Taizhou Hospital. The specimens were staged according to the 8th manual of the American Joint Committee on Cancer Staging Manual. The time distribution of patients undergoing lung surgery was from 2004 to 2012, and the clinical data and follow-up information of the patients were fully recorded. This study was approved by the Medical Ethics Committee of Taizhou Hospital, and the informed consent of all patients was obtained.

Immunohistochemistry (IHC)
Para n sections were baked in a 60°C in an incubator at 60°C for 2h before processing, and then taken out and cooled to room temperature. After depara nization and rehydration, the tissue sections were heated with citrate buffer to perform antigen retrieval in an autoclave. Then, 3% H 2 O 2 was added to the slices to block endogenous peroxidase. Normal sheep serum (Gibco) was used to block nonspeci c binding sites. The sections were incubated with anti-EGFL6 antibody (1:100, ab140079, Abcam, MA, USA) overnight. After that, the secondary antibody was added to the sections. Finally, the tissue sections were sequentially treated with hydrochloric acid, hematoxylin counterstain, dehydration and sealing.
These sections were graded by professional pathologists and were based on the staining intensity of and the percentage of positively stained cells. The intensity was divided into four grades: 0 (negative), 1 (weak), 2 (medium) and 3 (strong). The percentage of positive staining cells was graded as follows: 1 (0-25%), 2 (26-50%), 3 (51-75%), and 4 (76-100%). We multiplied the two scores, the nal score falling in the range of 0-6 was de ned as low EGFL6 expression, and 7-12 was de ned as high EGFL6 expression.

Cell culture
The human A549 LUAD cell line was purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). The authenticity of A549 cells (RRID: CVCL_0023) has been veri ed using Short Tandem Repeat (STR) pro ling within the last 3 years. A549 cells were cultured in RPMI 1640 medium containing 1% penicillin/streptomycin, 10% fetal bovine serum (FBS) (Gibco, Grand Island, NY, USA). Cells were maintained in an incubator at 37°C with a humidity of 5% CO 2 . All experiments were performed with cells without mycoplasma.

Cell transfection
The siRNA targeting EGFL6 (si-EGFL6) and the paired negative control (si-Ctrl) were synthesized by GenePharma (Shanghai, China). A549 cells were seeded in six-well plates and transfected with 5μl siRNA and 5μl Lipofectamine 2000 (Invitrogen). As for the overexpression of EGFL6, the LV-EGFL6-RNAi (sh-EGFL6) was obtained from GeneChem (Shanghai, China). A549 cells were grown in 24-well plates and transfected with lentivirus and 20μl HitransG according to the manufacturer's instructions.
Cell proliferation assay Cell Counting Kit-8 (CCK-8, Dojindo, Japan) was used to measure cell proliferation ability. The cells were cultured into 96-well plates, and 10μl CCK8 was added to each plate at 0, 24, 48, 72, 96h. Then, after 2h incubation at 37°C, the determination of cell proliferation was replaced by the absorbance value at 450nm.

Colony formation assay
The transfected cells were seeded in 6-well plates at a density of 1000 cells per well. After culturing for 10d, the colonies were xed with 4% paraformaldehyde for 30min and then stained with 0.1% crystal violet for 30min. The number of colonies was counted and compared in each group.
Migration and invasion assay A549 cells were transfected on the 24-well transwell plates (Corning Inc., Corning, NY). For migration assays, a serum free cell suspension (100μl) containing 5×10 4 cells was added into the upper chamber, and medium containing 10% FBS was added to the lower chamber. After incubating for 12h at 37°C, the cells at the bottom of the upper chamber were xed with 4% paraformaldehyde for 30min and stained with 0.1% crystal violet for 30min. Then, a cotton swab was used to remove the non-migrating cells in the upper chamber. For the invasion assay, the upper chamber was pre-coated with Matrigel (BD Biosciences, San Jose, CA), and the cell number of the cell suspension was changed to 1×10 5 . The remaining steps were the same as the migration assay. Finally, we counted and compared the number of cells of each group.

Wound healing assay
The wound healing assay was performed by culturing insert (ibidi GmbH, Martinsried, Germany) in 24well plates. The cell suspension (70μl) at a density of 5×10^4 cells/ml was placed into each well of the culture-insert. After incubating at 37°C and 5% CO2 for 24h, we used sterile tweezers to gently remove the culture-insert and photographed under an optical microscope. Then, we added serum free RPMI 1640 medium (300μl) to each well and cultured for 24h. Time lapse images were captured at the same position at 0 and 24h. Immunocytochemistry A cell suspension containing 1×10 4 cells was seeded on a coverslip in 12-well plates. After the cells were attached to the coverslips, they were xed with 4% paraformaldehyde for 30min. The cells were permeabilized in 0.5% Triton X-100 for 10min, then blocked with 10% FBS for 30min. The primary antibodies E-cadherin, N-cadherin and Vimentin (CST, Beverly, MA, USA; diluted 1:250) were used to incubate with the cells overnight at 4°C. On the next day, the cells were cultured with the uorescently labeled secondary antibody (diluted 1:250) for 1h in the dark. Later, the cells were stained with DAPI for 5min and then mounted. Images were captured under confocal microscope.
RNA isolation, reverse transcription and qPCR Total RNA was extracted from transfected A549 cells by TRIzol reagent (Invitrogen) following the manufacturer's instructions. Then, the total RNA was reverse transcribed into cDNA using the HiFiScript cDNA Synthesis Kit (CWBIO, Beijing, China). Quantitative real-time PCR analysis was performed by an ABI 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) with ChamQ Universal SYBR qPCR Master Mix (Vazyme, China) in triplicate. The GAPDH gene was utilized as housekeeping gene. All sequences were listed in Table S1.

Western blotting analysis
Total cellular protein of transfected cells was extracted using Radio-Immunoprecipitation Assay buffer (RIPA) containing protease inhibitor PMSF (100:1) and phosphatase inhibitor (Beyotime, Shanghai, China) (100:1). Quantitative of protein concentration was detected by BCA assay (Beyotime, Shanghai, China). The protein samples at the same quantity were separated by 10% SDS-PAGE and then transferred to PVDF membranes (Bio-Rad, Hercules, CA, USA). After blocking with Western Blocking Buffer (Biosharp, China) for 2h, the PVDF membranes were incubated with primary antibodies at 4°C overnight. The membranes were washed by TBS-Tween and incubated with HRP-conjugated secondary antibodies for 1h at room temperature. Chemiluminescent HRP Substrate (Millipore Corporation, Billerica, MA, USA) and ImageQuant LAS 500 (GE Health Care, Fair eld, CT, USA) were used to observe the immunoreactivity.
ImageJ software was utilized to quantify the intensity of the bands.

Quantitative analysis of medium EGFL6 level
An ELISA assay was used to verify that EGFL6 play a role through exocrine. The culture medium of the cells cultured for 2d was extracted and the concentration was detected by an ELISA kit (AMEKO, China). The optical density was tested at 450nm using the Multiskan FC Microplate Photometer (Thermo Fisher Scienti c, Waltham, MA, USA). The standard curve with OD value as the ordinate and standard concentration as the abscissa. The concentration of EGFL6 in each medium could be obtained by the measured OD value against the standard curve.

Osteoclast differentiation in vitro
BMMs were extracted from the femur and tibia of C57BL6 mice (8-week-old). After culturing for 4d or reaching 90% con uence in a 10cm cell culture dish, the BMMs were seeded into a 96-well plate at a density of 8000 cells/well in complete α-MEM containing 30ng/ml macrophage colonies stimulating factor (M-CSF). After 24h, we replaced the medium with complete α-MEM containing 30ng/ml M-CSF, 50ng/ml RANKL and supplemented with the culture medium of si-Ctrl, si-EGFL6, sh-Ctrl and sh-EGFL6 respectively. The osteoclast induction medium was changed every 2d until the 7th day. The cells were xed by 4% paraformaldehyde for 1h, and then stained by TRAP activity kit (Sigma-Aldrich, St. Louis, MO, USA). ImageJ software was used to quantify the area (percentage of a well) of TRAP-positive cells (≥3 nuclei).

Osteoblast differentiation in vitro
Bone marrow mesenchymal stem cells (BMSCs) were extracted from the femur and tibia of C57BL6 mice (8 weeks old), and cultured in complete α-MEM for 6d or reached 90% con uence in a 10cm cell culture dish. For osteoblast differentiation, BMSCs were re-seeded into a 12-well plate at a density of 1×10 4 cells/well with osteogenic medium (Complete DMEM supplemented with 10mM β-glycerophosphate, 50μM ascorbic acid and 100nM dexamethasone). The medium was changed every 2d. On day 7 and 21, the cells were stained with alkaline phosphatase (ALP), and the mineralized bone nodules were stained with Alizarin Red.

Bone absorption assay in vitro
Bone marrow mononuclear macrophage (BMMs) were seeded into 100μm bovine bone slices (Rongzhi Haida Biotech Co., Ltd, Beijing, China) at a density of 1×10^4 cells/well in α-MEM supplemented with 100 ng/ml RANKL and the culture medium of si-Ctrl, si-EGFL6, sh-Ctrl and sh-EGFL6 respectively. The osteoclast induction medium was changed every 2d until the 14th day. The cells were eliminated by mechanical agitation and sonication. Then, the resorption pits were examined by scanning electron microscope (Field Environmental Instruments Inc., Hillsboro, OR). ImageJ software was utilized to quantify the area of resorption pits.

Nude mouse tumor-induced bone destruction model in vivo
All animal experiments followed the guidance of the Institutional Animal Ethics Committee of Taizhou Hospital. 6-week-old female nude mice were randomly divided into si-Ctrl, si-EGFL6, sh-Ctrl and sh-EGFL6 group (6 mice/group). After induction of anesthesia, 20μl of transfected tumor cells (1×10^6 cells/ml) suspended in Phosphate Buffered Saline (PBS) were injected into the tibial marrow cavity through the tibial plateau of nude mice. All mice were euthanized on day 14. We removed the hind limbs of nude mice and compared the size of tumor. Then, the hind limbs were xed in 4% paraformaldehyde and collected for further experiments.

Histological and histomorphometric analysis
After decalci cation in 10% EDTA (ethylenediaminetetraacetic acid) for 14d, the xed hind limbs were embedded in para n and then sectioned. The tissue slices were utilized for H&E and TRAP staining. We acquired images of each sample through a high-quality microscope (Olympus, Japan) and counted the number of TRAP-positive cells in each sample.

Statistical analysis
The signi cant differences between groups were analyzed by Student's t-test or one-way ANOVA or Kruskal Wallis test using GraphPad Prism® version 8. 3

EGFL6 is elevated in lung adenocarcinoma and is associated with bone metastasis
We collected 30 random cases of LUAD samples and clinical data after surgery. IHC analysis was utilized to evaluate the expression of EGFL6 between LUAD tissues and adjacent normal tissues. The results showed that, in general, the expression of EGFL6 in LUAD tissues was higher than that in neighboring normal tissues (Fig. 1A, B). Through statistical analysis of clinical parameters, we found that the expression of EGFL6 was closely correlated with the TNM stage and bone metastasis of LUAD (Fig. 1C,  D). However, the expression of EGFL6 was not signi cantly related to the patients' age, histological grade, and lymph node metastasis ( Table 1). Effects of high and low expression of EGFL6 on the proliferation, migration and invasion of LUAD cells Through the analysis of clinical samples, we speculated that EGFL6 played a role in promoting the progression of LUAD. In order to explore the biological role of EGFL6 in LUAD, we used human LUAD cell line A549 cells for further study. A549 cells obtained overexpression (sh-EGFL6) and knockdown (si-EGFL6) phenotypes under lentivirus and siRNAs respectively. EGFL6-siRNA and null vector were applied to transfect A549 cells. WB and RT-PCR veri ed that the knockdown e ciency of EGFL6-siRNA-899 was the highest (Fig. S1A, B). the lentivirus was transfected into A549 cells and veri ed by uorescence (Fig.  S1C). Finally, we proved the knockdown and overexpression e ciency by WB and RT-PCR (Fig. S1D, E).
We performed CCK8 assays to test the relationship between the proliferation ability of A549 cells and the expression of EGFL6. The enhanced expression of EGFL6 signi cantly promoted the proliferation of A549 cells, while silencing EGFL6 weakened the viability of A549 cells ( Fig. 2A). Besides, the colony formation assays also showed the same trend with CCK-8 (Fig. 2B). In order to explore the migration and invasion abilities mediated by EGFL6, we conducted a transwell assay and a scratch test in vitro. As shown in Fig. 2C, the number of migrated and invading cells in EGFL6 overexpression group was signi cantly greater than that in the control group. On the contrary, the EGFL6 knockdown group had the opposite phenomenon. In addition, the cell scratch experiment found that the healing speed of the EGFL6 overexpression group was signi cantly faster than that of the control group, and depleting EGFL6 expression slowed the healing rate to an extent (Fig. 2D).
High expression of EGFL6 promotes EMT of lung adenocarcinoma cells EMT is an important biological process for LUAD cells to acquire the ability of migration and invasion. It  (Fig. S2). The WB results were consistent with RT-PCR results (Fig. 2E).
To further prove that EGFL6 can promote the EMT of A549 cells, we performed immunocytochemistry (ICC) to detect the expression of E-cadherin, N-cadherin and Vimentin in the si-Ctrl, si-EGFL6, sh-Ctrl and sh-EGFL6 groups. The results showed that the expression of N-cadherin and Vimentin in the sh-EGFL6 group was higher than that in the sh-Ctrl group, while the expression in the si-EGFL6 group was lower than that in the si-Ctrl group. The expression of E-cadherin was opposite to the above results (Fig. 3A).

EGFL6 partially regulates LUAD cells by activating Wnt and PI3K/AKT/mTOR signaling pathways
Wnt/β-catenin signaling pathway and PI3K/Akt/mTOR signaling pathway both play key roles in regulating the process of proliferation, migration and invasion of tumor cells. Through Western blotting, we observed that overexpression of EGFL6 increased the levels of GSK-3 and β-catenin compared to the control. Silencing EGFL6 signi cantly downregulated the levels of these signaling proteins (Fig. 3B). Moreover, the forced expression of EGFL6 increased the phosphorylation levels of PI3K, AKT and mTOR, while the low expression of EGFL6 did the opposite, with no difference in total protein level (Fig. 3C). Taken together, these evidences indicated that EGFL6 promoted the proliferation, migration and invasion of A549 cells at least in part through Wnt/β-catenin signaling pathway and PI3K/AKT/mTOR signaling pathway.

EGFL6 increases tumor growth and bone destruction in bone metastasis of LUAD cells in nude mice
To further validate the role of EGFL6 in promoting the migration and invasion of A549 cells, we established a bone metastasis model by implanting LUAD cells in nude mice. The suspensions of A549 cells of EGFL6 overexpression, knockdown, and their respective control were injected into nude mice through the tibial plateau. After 14d, all nude mice were euthanized, and the differences in tumor appearance size and bone destruction in each group were compared. The tumor growth rate of the sh-EGFL6 group was much faster than that of the sh-Ctrl group, while the si-EGFL6 group exhibited the opposite result (Fig. 4A).
In addition, we observed by X-ray that the sh-EGFL6 group had more severe tibial bone destruction than the sh-Ctrl group, while the si-EGFL6 group showed the opposite result (Fig. 4B). Then we performed para n sections on the tibia of nude mice and stained them with H&E and TRAP. H&E analysis supported that the osteolysis in sh-EGFL6 group was more severe than that of sh-Ctrl group. However, the opposite result was observed in the si-EGFL6 group compared with the si-Ctrl group (Fig. 4C). In addition, based on X-ray and H&E results, TRAP staining proved that osteoclasts in the bone surface were enhanced in the sh-EGFL6 group, but decreased in the si-EGFL6 group (Fig. 4D).
Since the results of animal models showed that the bone destruction in the EGFL6 overexpression group was more serious, in order to study the effect of EGFL6 on bone formation, we extracted the culture medium of each cell group and added them to the BMM and MC3T3 cells. Compared with the sh-Ctrl and si-Ctrl groups, BMM using sh-EGFL6 medium showed enhanced osteoclast differentiation (Fig. 4E), and MC3T3 using sh-EGFL6 medium had no signi cant change in osteogenic formation (Fig. 4F). According to the existence of the above-mentioned cell experiment, the in vitro bovine bone section resorption test showed that the bone resorption capacity of the sh-EGFL6 group was more potent than that of the control, and vice versa (Fig. 4G).
EGFL6 promotes osteoclast differentiation by activating NF-κB and downstream c-Fos/NFATc1 signaling pathways In order to clarify the potential mechanism of EGFL6 promoting osteoclast differentiation, we studied several classic signaling pathways in osteoclastogenesis by Western blotting. We rst divided BMMs into two groups. One group was treated with RANKL and a partial medium extracted from sh-EGFL6 (EGFL6 medium) to explore the levels of c-Fos and NFATc1. The other group was stimulated with RANKL and the medium extracted in sh-Ctrl for 0, 1, 3, and 5d. The results showed that under the stimulation of RANKL and control medium, the expression of c-Fos and NFATc1 reached the peak on the 5th day. In addition, when RANKL and EGFL6 medium were used together, the expression of c-Fos and NFATc1 was signi cantly enhanced on the 5th day (Fig. 5A).
Besides, we further studied the short-term signaling pathways of MAPKs, NF-κB and AKT under the in uence of EGFL6 medium and control medium, respectively. Under the pretreatment of EGFL6 medium or control medium, BMMs was induced by RANKL, and the phosphorylation of each protein in the MAPK, NF-κB and AKT signaling pathways was detected at 0, 5, 10, 20, 30 and 60min level. As shown in Fig. 5B, there was no signi cant difference in the activation and phosphorylation levels of AKT and MAPK members ERK, JNK and p38 between BMMs pretreated with EGFL6 medium and BMMs pretreated with control medium. In contrast, when induced by EGFL6 medium, the phosphorylation of NF-κB member IκBα increased at 10 min. Then, the phosphorylation of p65 in the trimeric structure was therefore similarly enhanced at 10min (Fig. 5C).

Discussion
It is well accepted that EGFL6 plays an important role in the formation of new blood vessels of epithelial cells, which is a necessary process for tumor growth and metastasis 11,16,19,20 . In this study, we found that EGFL6 expression was higher in LUAD tissues than that in adjacent normal tissues and EGFL6 staining in LUAD tissues was positively correlated with TNM stage and bone metastasis. Our results were consistent with a retrospective study of LUAD 17 , which indicated that high EGFL6 expression might serve as a marker of poor clinical outcome of LUAD, especially in younger patients 17 . In that study, the focus was mainly on the relationship between the expression level of EGFL6 and the prognosis of LUAD. Our study further discovered that the high expression level of EGFL6 in LUAD tissues was associated with bone metastasis. It has been proven that LUAD is the most common pathological type of lung cancer with a survival rate of 4-17% 21 . Compared with patients without bone metastases, the overall survival of LUAD patients with bone metastases was signi cantly shorter (5.8 months vs 10.2 months, P<0.05) 22 . Therefore, the poor prognosis in EGFL6-positive LUAD patients may be the result of enhanced bone metastasis. The expression of EGFL6 has also been shown to be positively correlated with the invasion, metastasis and poor prognosis of other cancers, such as ovarian cancer, breast cancer, colorectal cancer, gastric cancer, oral squamous cell carcinoma, nasopharyngeal carcinoma and benign meningioma [12][13][14][23][24][25][26] .
We also found that EGFL6 might promote bone metastasis of LUAD by stimulating bone destruction by osteoclasts. Bone is the preferred metastasis site for many tumor cells, such as breast, lung and prostate cancer cells [27][28][29] . We rst veri ed that EGFL6 was secreted to the culture medium by A549 cells. The medium containing a higher concentration of EGFL6 enhanced the osteoclast differentiation of BMMs.
The bone resorption assay also con rmed that EGFL6 signi cantly increased the area of bone resorption pits. Our in vivo experiments demonstrated that tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts were increased in the tibia of nude mice invaded by A549 cells with high expression of EGFL6. Moreover, studies have shown that when osteoblasts and bone metastatic breast cancer MDA-MB-231 cells were co-cultured, they can reduce the secretion of OPG and promote the secretion of monocyte chemotactic protein 1 (MCP1). This effect can be partially blocked by EGFR inhibitors 30 .
EGFL6 may promote bone metastasis and osteolytic lesions in the tumor microenvironment by decreasing OPG expression and increasing MCP1 expression in an EGFR-dependent manner 30 . In our study, the medium containing EGFL6 had little effect on the osteoblast differentiation of MC3T3 cells. In brief, our results suggest that the metastatic cells of LUAD may secret EGFL6 through paracrine, and stimulate bone resorption by stimulating osteoclast differentiation of BMMs.
Furthermore, we explored the underlying mechanism of EGFL6 promoting osteoclast differentiation. After the stimulation of RANKL in BMMs, NFATc1 and c-Fos, which play an essential role in the downstream pathways of osteoclast differentiation, were activated to control the cytokine expression 31,32 . We found that EGFL6 increased the expression of c-Fos and NFATc1 in a time-dependent manner. In addition, the MAPKs, NF-κB and AKT signaling pathways have been called the most classic signaling pathways in osteoclast formation. In the classic pathway of NF-κB, phosphorylation of IκBα induces the release of the RelA(p65)/p50 heterodimer, which causes nuclear translocation of p65 and ultimately leads to the activation of NF-κB 33 . In this study, we found that EGFL6 enhanced the phosphorylation of IκBα and subsequently the phosphorylation of p65, which means that EGFL6 promoted osteoclast differentiation in part by activating NF-κB and downstream c-Fos/NFATc1 signaling pathway. However, MAPKs members ERK, JNK, p38 and AKT were almost unaffected by EGFL6. It has been shown that EGF-like ligands are related to the bone environment. As a member of EGF-like ligands, EGFL6 is preferentially expressed in osteoblast-like cells, but has no effect on osteoblast proliferation and mineralization 11 . In addition, it is well known that osteoblasts are the main source of several cytokines responsible for regulating the differentiation and formation of osteoclasts, such as OPG, RANKL, M-CSF, and MCP1 34,35 .
Studies have shown that EGF-like ligands can stimulate the formation of TRAP-positive osteoclasts by indirectly reducing OPG expression and increasing MCP1 expression, which may contribute to the effect of metastatic breast cancer cells on bone 30 . However, the role of EGFL6 in bone metastasis of LUAD remains to be resolved.
Our in vitro studies have shown that knocking-in EGFL6 promoted the proliferation, migration and invasion of A549 cells, while silencing EGFL6 did the opposite. EMT is an important process for tumor cells to acquire the ability of migration, invasion and anti-apoptotic stimulation. EMT gives tumor cells the characteristics of metastasis, accompanied by the decrease of epithelial markers and the increase of mesenchymal markers 36 . Our study found that the mesenchymal markers N-cadherin and vimentin were signi cantly enhanced in knocking-in EGFL6 cells, but inhibited in EGFL6 silenced cells. This phenomenon indicated that EGFL6 promoted the ability of A549 cells to obtain migration and invasion and metastasize to bone, which might be partly through the enhancement of EMT. Besides, as a complex biological process of tumorigenesis and progression, EMT is regulated by a variety of signaling  [42][43][44][45] . Similarly, we found that knocking-in EGFL6 enhanced the expression of p-PI3K, p-AKT and p-mTOR in LUAD. The above results collectively proved that EGFL6 promoted the proliferation, migration and invasion of A549 cells at least in part through the enhanced EMT process and the activation of Wnt/β-catenin and PI3K/AKT/mTOR pathways. However, due to the characteristics of PI3K signaling pathway in regulating proliferation, migration and metastasis of tumor cells, the existing evidence was still insu cient to prove that EGFL6 drives the EMT of LUAD exactly through Wnt/β-catenin and PI3K/AKT/mTOR pathways. Further experiments remain to be carried out to explore the potential molecular mechanism of EGFL6 to promote LUAD metastasis.

Conclusion
Our ndings provide evidence that EGFL6 is highly expressed in LUAD tissues and is positively correlated with bone metastasis in LUAD. Knocking-in EGFL6 promotes osteoclast differentiation through NF-κB signaling pathway and promotes bone destruction in nude mice. As attested by the experiments in vivo and in vitro, we demonstrated that overexpression of EGFL6 signi cantly enhanced the proliferation, migration and invasion of A549 cells. On the contrary, the low expression of EGFL6 attenuated these capabilities. Further mechanism studies have found that EGFL6 promotes EMT, at least in part through the Wnt and PI3K/AKT/mTOR signaling pathways to promote the above-mentioned characteristics of A549 cells. Therefore, we speculate that EGFL6 tends to be a predictor of bone metastasis in LUAD. Availability of data and materials

Abbreviations
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests.

Con ict of interest
All authors declare no potential con ict of interest.    EGFL6 moderates the effect of A549 cells on skeleton system of nude mice and enhances the osteoclast differentiation of BMMs. a The tumor size in the sh-EGFL6 group is much larger than the sh-Ctrl group, and the si-EGFL6 group exhibited a smaller size than the si-Ctrl group. b Compared with each control, Xray shows that the bone destruction of tibia in sh-EGFL6 group is much severe while in sh-EGFL6 group is slight. c The tibial tumor sections of nude mice were stained with H&E. d The tibial tumor sections of nude mice were stained with TRAP. e EGFL6 enhances the osteoclast differentiation of BMMs. f EGFL6 has no signi cant in uence on osteoblast differentiation of MC3T3. g The bone resorption pits assay.

Supplementary Files
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Life Cycle and Transmission of Cyclospora cayetanensis: Knowns and Unknowns

Although infections with Cyclospora cayetanensis are prevalent worldwide, many aspects of this parasite’s life cycle and transmission remain unknown. Humans are the only known hosts of this parasite. Existing information on its endogenous development has been derived from histological examination of only a few biopsy specimens. Its asexual and sexual stages occur in biliary-intestinal epithelium. In histological sections, its stages are less than 10 μm, making definitive identification difficult. Asexual (schizonts) and sexual (gamonts) are located in epithelial cells. Male microgamonts have two flagella; female macrogametes contain wall-forming bodies. Oocysts are excreted in feces unsporulated. Sporulation occurs in the environment, but there are many unanswered questions concerning dissemination and survival of C. cayetanensis oocysts. Biologically and phylogenetically, C. cayetanensis closely resembles Eimeria spp. that parastize chickens; among them, E. acervulina most closely resembles C. cayetanensis in size. Here, we review known and unknown aspects of its life cycle and transmission and discuss the appropriateness of surrogates best capable of hastening progress in understanding its biology and developing mitigating strategies.

Introduction
Cyclosporiasis is a problem worldwide. Several recent papers review prevalence, clinical symptoms, diagnosis, epidemiology, and treatment of cyclosporiasis [1][2][3][4][5][6]. The objectives of this paper are to review known and unknown aspects of its life cycle and transmission, and to review the appropriateness of surrogates best capable of hastening progress in understanding its biology and developing mitigating strategies.

Taxonomy
Cyclospora cayetanensis is classified as a coccidian parasite, in the phylum Apicomplexa, family Eimeriidae. All members of Eimeriidae have a one-host, fecal-oral cycle. Eimeria species are among the most prevalent parasites of livestock and poultry; infections are so prevalent that it is very difficult to raise livestock free of Eimeria [12]. Eimeria are host-specific, and infections are generally confined to gastro-intestinal system. Indeed, Eimeria species causing poultry coccidiosis impose high costs to poultry production, worldwide. As with Cyclospora spp., oocysts of Eimeria are excreted in feces unsporulated (noninfectious), and sporulation occurs outside in the environment. Although Eimeria oocysts (encasing two sporocysts, each with four sporozoites) morphologically differ from those of Cyclospora (encasing two sporocysts, each with two sporozoites), they are phylogenetically related [13]. Cyclospora comprise a monophyletic group which share common ancestor with all species of Eimeria, to the exclusion of other extant taxa [13].

Life Cycle of C. cayetanensis
The life cycle of C. cayetanensis remains incompletely defined. The best-characterized stage is the oocyst. Humans are thought to be infected by ingesting food or water contaminated with oocysts ( Figure 1).  If a susceptible human ingests sporulated oocysts in contaminated food or water, the sporozoites inside the sporocysts excyst in the gut lumen and invade intestinal-biliary epithelial cells where the sporozoites transform into schizonts. Sexual multiplication occurs at the same site as asexual cycle. Microgamonts (male) contain flagellated microgemetes Microorganisms 2022, 10, 118 3 of 12 that fertilize macrogamonts to form the zygote. Oocysts then are excreted unsporulated in the feces. The prepatent period is thought to be around one week. Unsporulated oocysts are not infectious, they need to sporulate to became infective for a host. Under laboratory conditions, at 22 • C and 30 • C, sporulation will take around 7-14 days to occur outside the host [6].

Oocysts
The original description of C. cayetanensis elucidated most of the features presently understood about its oocysts [7]. Unsporulated oocysts in fresh fecal smears are spheroidal, 8-10 µm in diameter, with little or no size variation. The oocyst wall is colorless, thin (<1 µm), and bilayered. The inner layer encloses a central, undivided mass called the sporont; the sporont almost fills the oocyst and has many globules [7]. Sporulation occurs outside the host. Polar body and oocyst residuum are present. In fully-sporulated oocysts, there are two sporocysts, each with two sporozoites. Sporocysts are ovoidal,~4 × 6 µm, and contain Stieda and substieda bodies, and a large residuum. The Stieda bodies are plug-like structures involved in sporozoite excystation [12]. The sporozoites are elongated, 1 × 9 µm [7]. Whether C. cayetanensis sporozoites have crystalloid body or refractile bodies is not known. In Eimeria species, crystalloid bodies are considered virus-like particles; refractile bodies are proteinaceous in nature and are linked to host cell invasion [12].

Asexual Multiplication
After people ingest sporulated oocysts, sporozoites are believed (based on by analogy to more detailed data from various species of Eimeria) to excyst in the gut lumen and multiply, asexually, in intestinal epithelial cells. A few sporozoites may spill over and invade bile ducts and the gallbladder. Based on histological examination of endoscopic biopsies of duodenum or upper jejunum of Peruvian patients, asexual stages were observed in intestinal epithelial cells (presumably enterocytes) [10]. Only a few parasites were observed, and thus results were pioneering but preliminary. These stages were termed Types I and II meronts. Type I meronts each contained 8-12 small (3-4 µm long) merozoites; Type II meronts each contained four (12-15 µm) merozoites. However, the size of these merozoites was not confirmed by others [11]. The mode of division was not described.
Recently, an opportunity arose to study endogenous development of C. cayetanensis in an immunosuppressed cyclosporiasis patient with severe abdominal symptoms [11]. The entire gallbladder from this case had been surgically removed and fixed for histological and transmission electron microscopic (TEM) examination. In this gallbladder, profuse parasitization was observed; the diagnosis was confirmed by the presence of C. cayetanensis oocysts in feces and PCR, using DNA extracted from the gallbladder [11]. Cyclospora cayetanensis stages were found only in epithelial cells, located in a parasitophorous vacuole of host cytoplasm ( Figure 2). Mature schizonts were 7.6 × 5.1 µm and contained up to 10 merozoites. The size of merozoites could not be determined but appeared to be less than 6 µm long. By TEM examination, schizonts divided by schizogony (where the parasite nucleus divided into five or more nuclei before segregating into distinct merozoites). A residual body was present in some, but not in others. The number of generations of schizonts could not be determined because of overcrowding. By light microscopy, merozoites and schizonts in different phases of development appeared within the same host cell or in adjacent host cells. Whether the course of schizont development in this case was influenced by the immunosuppressive status of the patient could not be ascertained. Thus, caution is warranted before extrapolating; future studies should determine whether such asynchronous development occurs in immunocompetent patients. We are aware of no archived biopsy material available to support such studies.

Asexual Multiplication in Related Coccidia
Details of the asexual division process can aid diagnosis and illustrate the close affinity of C. cayetanensis with certain other coccidia. Considerable confusion surrounds the terminology used to describe asexual stages of coccidia. For the benefit of researchers not familiar with developmental cycles of coccidia, a brief account is provided here. Meront and schizont each describe stages undergoing asexual division. "Meront" has been used to denote various asexual stages, irrespective of division status [12]. Thus, it lacks perfect specificity. Some coccidia (for example, Toxoplasma gondii) replicate by "endodyogeny", wherein each mother cell produces, and is consumed by, exactly two daughter cells. Others (for example, species of Sarcocystis) divide by endopolygeny, where the mother cell's nucleus becomes a mass of connected lobes before subdividing into many daughter cells [12].
In species of Eimeria, schizonts divide by schizogony. After their sporozoites invade the host cell, the parasite nucleus divides into four or more nuclei before any merozoites are formed. After a defined number of multiplication cycles (termed generations), characteristic of each species of Eimeria, parasites from the final merozoite generation initiate the sexual cycle. The number of generations is fixed for each Eimeria species, and is not altered by immunosuppression [12]. Merozoites from each generation of schizonts are morphologically distinct.
Schizogony in C. cayetanensis resembles that in Eimeria. The lack of sequential specimens for study has, to date, precluded determination of the number of generations it undergoes prior to initiating the sexual cycle. Nonetheless, the occurrence of merozoites of different sizes suggests the existence of multiple generations of schizonts [12]. The development of merozoites by schizogony was confirmed by TEM examination. The presence of Type II merozoites could not be confirmed.
Cystoisospora belli is another coccidian that also parasitizes the small intestine and biliary system in humans [14,15]. In Cystoisospora, different generations of schizonts/Types can be found in the same host cell, because schizonts can rupture inside the host cell and merozoites can multiply within the same host cell without exiting the host cell ( Figure 3). Additionally, immature schizonts can retain the shape of merozoites (Figure 3). Whether this type of multiplication also occurs in C. cayetanensis remains to be determined. Because C. belli stages (including merozoites, gamonts and oocysts) are more than twice the size of their counterparts in C. cayetanensis, differential diagnosis is possible to the trained eye using light microscopy [15]. Genetic assays also support differential diagnosis [16][17][18].

Sexual Stages
Sexual stages of C. cayetanensis are smaller than 10 µm and occur in the same location as schizonts [11]. The male microgamonts measure 6.6 × 5.2 µm and contain fewer than 20 microgametes organized around a residual body. Each microgamete measures up to 2 µm long and is flagellated. Female macrogametes contain distinctive eosinophilic wall-forming bodies that vary in size and are less than 1 µm in HE-stained sections. Macrogamonts measure 5.8-6.5 × 5.3-6.5 µm. Oocysts in sections are unsporulated and have a diameter of 5.7-7.5 µm. The TEM examination confirmed the histologic findings [11].

Need for Surrogates for Studying C. cayetanensis
Only rarely have scientists been positioned to directly study C. cayatanensis infections in vivo. Even access to oocysts is limited, undermining efforts to understand their maturation and senescence. What we know of human pathology and the parasite's development in human hosts has been derived from just a few case studies. Ethical constraints essentially preclude the ability to examine infections under defined or replicated conditions, and the absence of any in vitro or animal model for studying this organism further impede progress in identifying vulnerabilities that might be exploited to strengthen prevention or cure.
Fortunately, a wealth of data from natural and experimental Eimeria infections provide helpful context for interpreting what scant evidence we do have (see above). Furthermore, Eimeria can and should be exploited as surrogates for evaluating methods requiring large numbers of C. cayetanensis oocysts. For example, maturation of oocysts of Eimeria acervulina (a common poultry parasite) entails upregulation of a suite of genes most of which have homologues in C. cayetanensis [19]. These abundant parasites, which pose no risk to the health of human investigators, can speed efforts to evaluate methods to filter parasites from irrigation water, and treat food in ways that may render such contaminants harmless, or treat infections when prevention fails.

Molecular Tools and Comparative Genomics
Below, we briefly review insights from comparative genetics and genomics of Cyclospora illustrating the potential, and limits, of related organisms as surrogates to inform us of opportunities to better understand, and manage, this elusive but important human enteric pathogen .

Small-Subunit rDNA Sequence
Molecular tools have revolutionized the exploration of biodiversity. Sequencing nuclear-encoded small-subunit rDNA gene (18S) has especially aided reconstruction of relationships among organisms for which morphological taxonomy is challenging [20]. Comparisons of SSU-rRNA sequences between human and primate isolates of Cyclospora spp. identified 98% similarity in that gene [21], validating their distinctness but testifying to their close evolutionary relationship. SSU-rDNA was also utilized to understand the relatedness among closely related species. Strikingly, examination of SSU-rRNA sequences revealed a 94 to 98% sequence similarity between Cyclospora and Eimeria species. The neighbor-joining tree constructed using 18S rDNA sequences suggests C. cayetanensis is most closely related to Eimeria species that infect birds ( Figure 4A). Thus, molecular analyses of nuclear SSU rDNA sequences suggest that C. cayetanensis is closely related to other coccidia, especially members of the genus Eimeria in the family Eimeriidae.  This molecule serves as the target for FDA-validated assays for diagnosing contamination of certain types of fresh produce with C. cayetanensis from fresh produce and is also used to diagnose infections in clinical samples [22][23][24][25]. The sensitivity of assays targeting this gene benefits from the fact that each genome of the parasite encodes multiple copies of the SSU rRNA. Recently, a real-time qPCR assay targeting the C. cayetanensis 18S rDNA gene has been optimized to improve detection sensitivity, as validated in the FDA Bacteriological Analytical Manual (BAM) for C. cayetanensis testing [24].

Organellar Genomes
Organellar genomes, residing in the mitochondrion and apicoplast, illuminate relatedness among closely related apicomplexan species because they are inherited maternally without genetic recombination [26,27]. Recently, the mitochondrial and apicoplast genomes of C. cayetanensis have been sequenced and compared with closely related Eimeria species [28][29][30][31]. As with other coccidian parasites, the mitochondrial genome of C. cayetanensis is 6229 bp in size with 33% GC content [28][29][30][31]. The mitochondrial genome is organized in a linear molecule with approximately 513 copies; these are arranged in concatemeric structures with a head-tail configuration, as in other Eimeria mitochondrial genomes. As with other coccidian parasites, the mitochondrial genome of C. cayetanensis encodes three protein-coding genes (cox1, cox3, and cytB), in addition to 14 large subunit (LSU) and nine small subunit (SSU) fragmented rRNA genes [30]. Comparing the amino acids comprising the mitochondrially-encoded proteins of C. cayatanensis to those of closelyrelated species of Eimeria documented sequence similarities ranging from 90-97% for cytbB, 93-97% for cox1, and 83-93% for cox3 [30]. Additionally, comparative phylogenomic analysis of mitochondrial genomes also revealed the monophyletic clustering of C. cayetanensis with the rabbit-infecting parasite E. magna ( Figure 4B) [30]. Comparing the mitochondrial genomes of isolates of C. cayetanensis identified 8 single nucleotide polymorphisms and one 7-bp multiple-nucleotide in the junction area between genome copies [28,32].
The apicoplast of C. cayetanensis, a non-photosynthetic plastid derived from a secondary symbiosis, is 34,155 bp in length, with a GC content of 22%, and harbors 29 proteincoding genes, 33 tRNA, and four rRNA genes [28,33]. Genes are encoded bidirectionally. A recent comparative study of sequence variation of apicoplast genomes of C. cayetanensis identified 25 single nucleotide differences amongst 11 isolates from several geographical locations, in addition to a unique 30 bp-long repeat insertion sequence in a Nepalese sample [28,33].
Comparing the apicoplast genome of C. cayetanensis to that of Eimeria tenella (Table 1) revealed complete conservation of gene order (synteny) and strong (85.6%) sequence conservation ( Figure 4C) [28]. Thus, the genetic similarity between Eimeria species and C. cayetanensis in each organellar genomes substantiate avian Eimeria as a sensible surrogate to study drug treatment, and therapeutics specifically targeting the organellar genomes.

Nuclear Genome
Currently, 39 nuclear genomes of C. cayetanensis have been assembled, using either short paired-end reads from Illumina sequencing (Illumina, San Diego, CA, USA) or Roche GS-FLX 454 sequencing (454 Life Sciences, Branford, CT, USA, available online at https://www.ncbi.nlm.nih.gov/assembly/?term=cyclospora, accessed on 15 November 2021) [34,35]. Of these 39 genomes, CcayRef3 (GCF_002999335) and CHN_HEN01 (GCF_000769155) presently serve as the reference genomes of C. cayetanensis. The total nuclear genome size is estimated to be~44 Mb (Table 1) [34,35]. These genomes have roughly similar GC composition (52%) and are predicted to encode~7500 genes; they have been resolved to 738 contigs (Table 1). Not surprisingly, comparative genome analysis revealed that the genome organization, metabolic capabilities, and potential invasion mechanisms are very similar to those of E. tenella [34]. When compared with more distantly related relatives that form tissue-cysts (such as T. gondii), these monoxenous parasites are notably reduced in rhoptry protein kinases, phosphatases, and serine protease inhibitors. This seems significant because of the dominance of such protein domains in tissue-cyst forming coccidian parasites; this underscores the special suitability of Eimeria as surrogates to study the enigmatic Cyclospora spp. Note, however, that even these close relatives differ, reminding us of the uniqueness of each biological species: a family of TA4-type surface antigens (SAG), highly abundant in E. tenella, have not been detected in C. cayetanensis [34]. In addition, plant-like AP2 transcription factors are slightly more abundant in C. cayetanensis than in E. tenella, consistent with these being the major transcription factors in apicomplexans [34].

Developmentally-Regulated Genes
Genome assembly and annotation for Cyclospora and for its Eimeria relatives remain far from complete, impeding efforts to understand their comparative biology. Nonetheless, a recent study of sporulation in E. acervulina suggests that similar genes in each species may be important regulators or effectors of parasite maturation [19]. Maturing cohorts of E. acervulina undergo concerted changes in the expression of many genes; most of these increase their expression over the course of sporulation. Genes strongly expressed throughout sporulation included ATPases (like EAH 4100 and EAH 4110) and oocyst wall proteins (like EAH 33530). Members assigned to the SAG family of surface antigens (EAH 59200, 59950, and 11680) were expressed to a much greater extent in mature than in immature oocysts. Although the function of many other genes remains unknown, guided at best by predictions from gene ontology, it is notable that about two-thirds of these genes have strongly conserved genes in C. cayetanensis. Until it becomes possible to propagate and study C. cayetanensis (in animal models, in vitro, or in organoid cultures), Eimeria surrogates offer a biologically sensible means to experiment with parasites, to define their biology, tolerances, and vulnerabilities.

Conclusions
To counter the harms posed by C. cayetanensis, producers, public health authorities, clinicians and researchers require more empirical studies of this enigmatic parasite. However, the episodic nature of its transmission and the absence of in vitro or in vivo means to propagate the parasite severely limit such opportunities. Fortunately, a wealth of information exists for the veterinary parasites which Cyclospora most closely resembles. These similarities span transmission, development, genetics, pathogenesis, and molecular biology. The experimental opportunities afforded by these readily studied surrogates should hasten progress in understanding, and better managing, threats to food safety and public health.


extfieldsofstudy: Chemistry Medicine

s2fieldsofstudy: Biology Chemistry

text:
Protein Phosphatase 2A Inhibits Gastric Cancer Cell Glycolysis by Reducing MYC Signaling

Background: The Warburg effect is closely associated malignant phenotypes and poor prognosis in cancer patients. PP2A is a highly conserved eukaryotic serine/threonine protein phosphatase that functions as a tumor suppressor in a variety of human cancers. However, the relationship between PP2A and the Warburg effect has yet to be fully understood. Methods: The expression prole of two endogenous inhibitors of PP2A, SET and CIP2A, are detected by real-time qPCR. Loss-of-function and gain-of-function are performed to demonstrate the roles of PP2A in gastric cancer cell proliferation and glycolysis. Cell biological, molecular, and biochemical approaches are used to uncover the underlying mechanism. Results: In this study, we nd that SET and CIP2A are overexpressed in gastric cancer and associates a decreased PP2A activity. Pharmacological activation of PP2A with FTY-720 and DT-061 signicantly reduces gastric cancer cell proliferation and glycolytic ability. Importantly, inhibition of PP2A activity by genetic silencing of PPP2R5A induces a growth advantage, which can be largely compromised by addition of the glycolysis inhibitor 2-Deoxy-D-glucose, suggesting a glycolysis-dependent effect of PP2A in gastric cancer. Mechanistically, the well known transcription factor and glycolysis regulator c-Myc is discovered as the functional mediator of PP2A in regulating cell glycolysis. Ectopic expression of a phosphorylation-mutant c-Myc resistant to PP2A (MycT58A) restores the inhibitory effect of FTY-720 and DT-061 on the lactate production and glucose uptake. Furthermore, there is a close association between SET and CIP2A expression and c-Myc gene signatures in gastric cancer samples. Conclusions: This study provides strong evidence of the involvement of PP2A in the Warburg effect and indicates that it could be a novel antitumor strategy to target tumor metabolism in gastric cancer.

Introduction
Gastric cancer is one of the most common malignant tumors in the world. The incidence and mortality of gastric cancer in East Asia and China remain high [1,2]. Due to the low frequency of regular gastroscopy, insidious early symptoms, poor results of radiotherapy and chemotherapy, the mortality rate of gastric cancer has remained high, the clinical outcome of patients with advanced and metastatic gastric cancer is still not optimistic [3]. Therefore, in-depth studying of the molecular mechanism involved in gastric cancer occurrence and development and developing effective diagnosis and treatment methods are urgently needed.
Due to the hypoxia and low pH microenvironment, the glucose metabolism of tumor cells is different from that of normal cells, which is manifested by high glycolysis but not oxidative phosphorylation and known as the Warburg effect [4,5]. Abnormal energy metabolism especially the Warburg effect is an emerged as a key feature of most tumor cells [6]. Increased Warburg effect is associated many oncogenic phenotypes in cancers, such as rapid cell proliferation, drug resistance, and stemness [7]. Therapies targeting the Warburg effect have a profound inhibitory effect on tumor progression. In gastric cancer, glycolytic pro ciency negatively affects survival outcomes of metastatic gastric cancer patients treated with paclitaxel-ramucirumab systemic therapy [8]. Notably, several reports have documented the regulators of the Warburg effect in gastric cancer, such as MACC1 [9], FBP1 [10], WTAP [11], and MUC16 [12]. However, the molecular mechanisms underlying the Warburg effect in gastric cancer remain unclear.
Protein Phosphatase 2A (PP2A) is a highly conserved eukaryotic serine/threonine protein phosphatase with functions that counter-balance kinase-mediated phosphorylation [13]. PP2A is a heterotrimeric enzyme comprised of a scaffolding subunit (A), regulatory subunit (B) and a catalytic subunit (C) [14].
PP2A is frequently inactivated in human cancers and is considered as a tumor suppressor. Therefore, strategies of improving PP2A activity have been regarded as a promising therapeutic intervention. PP2A activity is tightly regulated by several endogenous inhibitors especially SET and cancerous inhibitor of PP2A (CIP2A), which have been shown to be overexpressed in a variety of human cancers [15]. SET is rst identi ed as inhibitor of PP2A through isolation from bovine kidney and is upregulated in many cancers such as leukemia and breast cancer [16]. CIP2A is an oncoprotein that prevents PP2A-mediated dephosphorylation of c-Myc at Ser 62 and contributes to the stabilization of c-Myc [15]. PP2A has been shown to regulate many cellular processes in gastric cancer including, but not limited to, growth, stemness, and apoptosis [17,18,19,20]. However, the link between PP2A and the Warburg effect remains largely unknown.
In this study, we show that highly expressed SET and CIP2A lead to reduced PP2A activity in gastric cancer. Pharmacological activation of PP2A with FTY-720 or DT-061 decreases the colony formation ability and glycolytic capacity of gastric cancer cells. PP2A activation decreases phosphorylated c-Myc levels and c-Myc transcriptional activity. Taken together, our study suggests that PP2A plays an important role in the Warburg effect by modulating c-Myc signaling.

Gene expression analysis
For analysis of SET and CIP2A expression in gastric cancer samples, data from The Cancer Genome Atlas (TCGA) and the Genotype-Tissue Expression (GTEx) were used and analyzed by the GEPIA database [21]. TCGA RNA-seq data of gastric cancer samples (n = 408) was used for correlation analysis and the correlation coe cient was determined by Spearman method.
Scramble non-target siRNAs were used as negative controls. Cell transfection was performed with 15 µmol siRNAs by Lipofectamine RNAiMAX reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. Western blotting was used to determine knockdown e ciency.

Detection of PP2A activity
The PP2A activity in response to FTY-720, DT061, and PPP2R5A knockdown was detected by PP2A Immunoprecipitation Phosphatase Assay Kit (Millipore, USA) in according to the manufacturer's protocol. Brie y, gastric cancer cells with indicated treatment were lysated by NP-40 lysis buffer supplemented with protease inhibitor cocktail. Then, cell lysates was mixed with PP2A antibody and protein A slurry. After washing with TBS and assay buffer for three times, phosphopeptide was added and allowed to incubation for 10 min at room temperature. Finally, Malachite green solution was added, followed by absorbance detection at 650 nm.

Western blotting
Gastric cancer cells with indicated treatment were rinsed with cold PBS before treated with RIPA lysis buffer (50 mM Tris, 0.15 M NaCl, 1 mM EGTA, 1% NP40, 0.25% SDS, pH 7.4) containing protease and phosphatase inhibitors. Cell pellets were incubated for 30 min on ice to homogenize fully and then total proteins in the supernatant of cell lysates were collected by centrifuging at 4°C at 12,000 rpm for 10 min.

Glucose uptake and lactate production
Glucose and lactate levels in the culture medium were detected using Glucose Assay kit (Biovision, Milpitas, CA, USA) and Lactate Assay kit (Biovision, Milpitas, CA, USA), respectively. In brief, gastric cancer cells were seeded at 5 x 10 5 cells in 6-cm cell culture dishes and allowed to grow for 24 h with indicated treatment. After 24 h, the culture medium collected and centrifuged at 2,000 rpm for 5 min to obtain the supernatant without cell debris. Finally, glucose and lactate assays were performed according to the manufacturer's instructions, and colorimetric density was assessed using a Multifunctional microplate reader.

Plate colony formation assay
Gastric cancer cells with were resuspended and plated in 6-well plates at a density of 500 cells per well and allowed to grow for 10-14 days with indicated treatment. Then the cells were xed with 4% paraformaldehyde for 15 min at room temperature and stained with 0.5% crystal violet for 15 min. The experiment was performed in triplicate.

Measurement of c-Myc transcriptional activity
The c-Myc transcription factor assay Kit (Abcam, ab207200) was used to quantify c-Myc activation in nuclear extracts from gastric cancer cells. In brief, HGC-27 and MGC-803 cells were subjected to treatment with 5 µM FTY-720 or 10 µM DT-061 for 24h, followed by detection of c-Myc transcriptional activity in according to the manufacturer's instructions.

Statistical analysis
All data were represented as mean ± standard deviation (SD). GraphPad 6.0 (GraphPad Software Inc., San Diego, CA) was used for statistical analysis. The correlation of gene expression was evaluated by Spearman's correlation. P-values were calculated by two-tailed unpaired Student's t-test or one-way ANOVA. A value of P < 0.05 was considered to be statistically signi cant.

Results
3.1. Increased SET and CIP2A expression are associated with reduced PP2A activity in gastric cancer To begin investigating the role of PP2A in gastric cancer, we rst determined the expression of SET and CIP2A, two known oncogenic inhibitors of PP2A. Consistent with previous reports, data obtained from TCGA and GTEx cohorts showed that SET and CIP2A expression levels were high in gastric cancer tissues compared with normal samples (Fig. 1A). By real-time qPCR analysis, we found that SET and CIP2A mRNA expression were signi cantly increased in 87.5% (7/8) and 75% (6/8) of the gastric cancer cell lines, respectively (Fig. 1B). By measuring the PP2A activity, we found that 75% (6/8) of the gastric cancer cell lines had an increased PP2A activity compared with the nonmalignant GES1 cells (Fig. 1C). Of note, SET or CIP2A expression was closely associated with a reduced PP2A activity in gastric cancer cell lines (Fig. 1D). Therefore, decreased PP2A activity in gastric cancer appears to be induced by aberrant SET and CIP2A expression.
3.2. FTY-720 and DT-061 suppress gastric cancer proliferation in a dose-dependent manner To better understand the role of PP2A in gastric cancer, we used a known SET inhibitor FTY-720 that activates PP2A and a small-molecule activator of PP2A DT-061 to treat HGC-27 and MGC-803 cells [22]. As shown in Fig. 2A

PP2A activation inhibits the Warburg effect in gastric cancer
Occasionally, we found that cell culture medium was acidi ed much slower after FTY-720 and DT-061 treatment for 24 h. Therefore, we speculated a potential role of PP2A in regulating lactate production, a classical character of the Warburg effect. To test this hypothesis, we measured the lactate levels in the culture medium of HGC-27 and MGC-803 cells after treatment with different concentrations of FTY-720 and DT-061. After normalization with total cell protein, we observed that the lactate level was signi cantly reduced by FTY-720 in HGC-27 and MGC-803 cells in a dose-dependent manner (Fig. 3A). Likewise, similar effects were also induced by DT-061 (Fig. 3B). To further con rm the impact of PP2A on the Warburg effect, we tested the glucose uptake upon FTY-720 and DT-061 treatment in gastric cancer cells. As a result, both FTY-720 and DT-061 resulted in a marked reduction of glucose uptake in HGC-27 and MGC-803 cells (Fig. 3C-D). Moreover, using real-time qPCR, we found that glycolytic genes including HK2, PFKL and LDHA were also signi cantly downregulated by FTY-720 or DT-061 treatment (Fig. 3E). Consistently, correlation analysis revealed that there was a high correlation between SET and CIP2A expression and glycolytic genes (HK2, PFKL and LDHA) in gastric cancer samples (Fig. 3F).

Growth advantage induced by PP2A inhibition is glycolysis-dependent in gastric cancer
Because that PP2A can inhibit gastric cancer proliferation and reduce the glycolytic ability, we next tested whether PP2A affect tumor growth via modulation tumor glycolysis. To address this hypothesis, we rst genetically silenced PPP2R5A, a regulatory B subunit of the major PP2A protein complex [23], in two gastric cancer cell lines, MKN28 and SGC-7901. The knockdown e ciency was shown in Fig. 4A. PPP2R5A knockdown led to signi cant decrease in PP2A activity (Fig. 4B). Interestingly, lactate production (Fig. 4C) and glucose uptake (Fig. 4D) of MKN28 and SGC-7901 were remarkably upregulated by PPP2R5A knockdown. Moreover, plate colony formation assay showed that PPP2R5A knockdown boosted cell proliferation of MKN28 and SGC-7901 cells, which can be largely abrogated by addition of the known glycolysis inhibitor 2-DG (Fig. 4E). Collectively, these data above suggest that PP2A couples cell glycolysis to tumor cell proliferation in gastric cancer.
3.5. PP2A modulates c-Myc expression to suppress gastric cancer glycolysis PP2A complex dephosphorylates serine 62 (S62) of c-Myc, rendering c-Myc as a substrate for FBXW7mediated ubiquitination and subsequent degradation by the 26S proteasome. Importantly, c-Myc is a known transcriptional factor of the Warburg effect [24]. Therefore, we reasoned that PP2A targets c-Myc to modulate gastric cancer cell glycolysis. By western blotting, we found that PP2A activation by FTY-720 or DT-061 decreased the S62-phosphorylated c-Myc and total c-Myc levels in HGC-27 and MGC-803 cells (Fig. 5A). As the second line of evidence, c-Myc transcriptional activity was also suppressed by FTY-720 and DT-061 treatment (Fig. 5B). To determine whether aberrant expression of c-Myc can restore the suppressive effect induced by PP2A activation, we ectopic introduced MycT58A, a phosphorylationmutant resistant to PP2A in HGC-27 and MGC-803 cells. As shown in Fig. 5C, the inhibitory effect of FTY-720 and DT-061 treatment on the lactate production and glucose uptake was largely restored by MycT58A. Furthermore, correlation analysis showed that SET or CIP2A expression was positively associated with c-Myc gene signature in gastric cancer samples (Fig. 5D). Collectively, c-Myc is a critical mediator for PP2A-induced suppressive effects on cell proliferation and glycolysis in gastric cancer (Fig. 5E).

Discussion
PP2A plays diverse roles in human cancers [22,25,26]. In this study, we investigated (i) the expression pattern of two cellular inhibitors of PP2A, SET and CIP2A, in gastric cancer, (ii) the effect of PP2A activation on tumorigenic potential and the Warburg effect, (iii) whether increased growth advantage induced by PP2A inhibition is glycolysis-dependent, (iv) the underlying molecular mechanism by which PP2A regulates the Warburg effect. Our results suggest that PP2A is profoundly implicated in the Warburg effect and be exploited as potential targets for gastric cancer therapy.
It is well documented that increased expression of PP2A-inhibitory proteins such as SET and CIP2A contributes to decreased PP2A activity in cancers [27]. SET protein levels are highly expressed in various human tumors, including chronic myeloid leukemia, pancreatic cancer, and colorectal cancer; in gastric cancer, SET is reported to maintain cancer cell stemness by suppressing PP2A activity and stabilizing E2F1 protein [28]. Moreover, CIP2A is overexpressed in gastric cancer and predicts a poor prognosis; CIP2A knockdown is shown to negatively affect clonogenicity and senescence of tumor cells [29,30,31]. In this report, we con rmed that SET and CIP2A expression was highly expressed in gastric cancer samples and their dysregulation led to reduced PP2A activity.
SET can be targeted by FTY-720 (Fingolimod), a sphingosine analogue, is an FDA approved immunosuppressant used for the treatment of multiple sclerosis [13]. FTY720 is able to disrupt the interaction between SET and PP2A, thus resulting in elevated PP2A activity. FTY720 has been reported to increase the expression of Cip1/p21, p27, and BH3-only proteins to induce gastric cancer cell apoptosis and has an additive effect in killing cancer cells when in combination with Cisplatin [32]. Consistently, we revealed that FTY720 suppressed gastric cancer cell proliferation in a dose-dependent manner. Moreover, activation of PP2A with DT-061, a phenothiazine derivative, induced similar tumor-suppressive effects in gastric cancer cells. Apart from previous reports regarding the roles of PP2A in gastric cancer, we for the rst time identi ed a novel link between PP2A and the Warburg effect. FTY720 and DT-061 treatment signi cantly boosted the Warburg effect of gastric cancer cells as evidenced by increased lactate release, glucose uptake, and expression of glycolytic genes. Moreover, PP2A inactivation by knockdown of PPP2R5A facilitated cell growth and blocking glycolysis with 2-DG largely abrogated this effect. Therefore, our ndings further broaden the roles of PP2A in gastric cancer and suggest a glycolysisdependent effect of PP2A.
Prior works have highlighted c-Myc as critical substrate of PP2A complex in cancers [25,33,34,35]. For instance, PP2A activation by a novel SET antagonist OP449 decreases S62 phosphorylation of c-Myc and reduces c-Myc activity and expression of downstream target genes in breast cancer [36]. In MYCdriven cancer, DT-061 treatment inhibited c-Myc expression via proteasome-mediated degradation, resulting in tumor growth inhibition [37]. Given the important role of c-Myc in regulating the Warburg effect, we also tested c-Myc changes in the presence of PP2A activation. Expectedly, S62 phosphorylation of c-Myc was reduced by FTY720 or DT-061 treatment in gastric cancer cells and ectopic expression of c-MycT58A was competent to restore the inhibitory effect of PP2A on the Warburg effect. However, future work comprehensively illustrating the full spectrum of substrates of PP2A and whether these substrates are involved in the regulation of the Warburg effect may be very important.

Conclusion
In conclusion, our results reveal that dysregulation of PP2A-inhibitory proteins leads to reduced PP2A activity. We propose that PP2A regulates c-Myc activity to transcriptionally repress the expression of glycolytic genes and the Warburg effect. Given the important role of the Warburg effect in facilitating tumor growth, our data further supports the pursuit of PP2A as a target for cancer therapy in gastric cancer.

Availability of data and material
All data needed to evaluate the conclusions in the paper are present in the paper. Additional data related to this paper may be requested from the authors.

Competing interests
The authors declare that they have no competing interests.